The cyclic tetradecapeptide somatostatin-14 (SRIF) was originally isolated from the hypothalamus and characterized as a physiological inhibitor of growth hormone release from the anterior pituitary. It was characterized by Guillemin et al. and is described in U.S. Pat. No. 3,904,594 as having the amino acid sequence: (cyclo 3-14)H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (SEQ ID NO:1). This tetradecapeptide has a bridging or cyclizing bond between the sulfhydryl groups of the two cysteinyl amino acid residues in the 3- and 14-positions. SRIF was found to also regulate insulin, glucagon and amylase secretion from the pancreas, and gastric acid release in the stomach, e.g. it inhibits the effects of pentagastrin and histamine on the gastric mucosa. SRIF is also expressed in intrahypothalamic regions of the brain and has a role in the regulation of locomotor activity and cognitive functions. SRIF is localized throughout the central nervous system, where it acts as a neurotransmitter. In the central nervous system, SRIF has been shown to both positively and negatively regulate neuronal firing, to affect the release of other neurotransmitters, and to modulate motor activity and cognitive processes.
Somatostatin and many analogs of somatostatin exhibit activity in respect to the inhibition of growth hormone (GH) secretion from cultured, dispersed rat anterior pituitary cells in vitro; they also inhibit GH, insulin and glucagon secretion in vivo in the rat and in other mammals. One such analog is [D-Trp8]-SRIF, which is disclosed in U.S. Pat. No. 4,372,884. Somatostatin has also been found to inhibit the secretion of gastrin and secretin by acting directly upon the secretory elements of the stomach and pancreas, respectively, and somatostatin is being sold commercially in Europe for the treatment of ulcer patients. The powerful inhibitory effects of somatostatin on the secretion not only of GH but also of insulin and glucagon have led to studies of a possible role of somatostatin in the management or treatment of juvenile diabetes and have proved useful in studying the physiological and pathological effects of these hormones on human metabolism. SRIF is also known to inhibit the growth of certain tumors.
SRIF affects multiple cellular processes. Studies have shown that SRIF is an inhibitory regulator of adenylyl cyclase in different tissues. SRIF also regulates the conductance of ionic channels, including both K+ and Ca2+ channels. These actions of SRIF are mediated via pertussis toxin-sensitive guanine nucleotide-binding proteins. SRIF also regulates the activity of tyrosine phosphatases, the Na+/H+ antiport, and cellular proliferation through pertussis toxin-insensitive mechanisms.
SRIF induces its biological effects by interacting with a family of membrane-bound structurally similar receptors. Five SRIF receptors have been cloned and are referred to as SSTR1-5. Human SSTR1, mouse SSTR2 and mouse SSTR3 are described in Raynor et al., Molecular Pharmacology, 43, 838-844 (1993), and all five human SRIF receptors are now available for research purposes. Human SSTR1, 2 and 3 are also disclosed in U.S. Pat. No. 5,436,155. Additional SRIF receptors are disclosed in U.S. Pat. Nos. 5,668,006 and 5,929,209. All five receptors bind SRIF and SRIF-28 with high affinity. Selective agonists at SSTR2 and SSTR5 have been identified and used to reveal distinct functions of these receptors. These two receptors are believed to be the predominant subtypes in peripheral tissues. SSTR2 is believed to mediate the inhibition of growth hormone, glucagon and gastric acid secretion. In contrast, SSTR5 appears to be primarily involved in the control of insulin and amylase release. SSTR3 mediates inhibition of gastric smooth muscle contraction. SSTR4 is found in the pituitary, lungs, GI tract, kidneys, and in certain tumors to the substantial exclusion of the other SRIF receptors; it is believed to be activated upon binding by SRIF. These overall findings indicate that different receptor subtypes mediate distinct functions of SRIF in the body. Additional functions of SSTR4 could be learned if a highly selective agonist or antagonist was available.
There are different types of tissues in the human body that express somatostatin receptors including: (1) the gastrointestinal tract, likely including the mucosa and smooth muscle, (2) the peripheral nervous system, (3) the endocrine system, (4) the vascular system and (5) lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogs.
Somatostatin receptors are also expressed in pathological states, particularly in neuroendocrine tumors of the gastrointestinal tract. Most human tumors originating from the somatostatin target tissue have conserved their somatostatin receptors. It was first observed in growth hormone producing adenomas and TSH-producing adenomas; about one-half of endocrine inactive adenomas display somatostatin receptors. Ninety percent of the cardinoids and a majority of islet-cell carcinomas, including their metastasis, usually have a high density of somatostatin receptors. However, only 10 percent of colorectal carcinomas and none of the exocrine pancreatic carcinomas contain somatostatin receptors. The somatostatin receptors in tumors can be identified using in vitro binding methods or using in vivo imaging techniques; the latter allow the precise localization of the tumors and their metastasis in the patients. Because somatostatin receptors in gastroenteropancreatic tumors are functional, their identification can be used is to assess the therapeutic efficacy of an analog to inhibit excessive hormone release in the patients.
A cyclic SRIF analog, variously termed SMS-201-995 and Octreotide, i.e. D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol is being used clinically to inhibit certain tumor growth; analogs complexed with 111In or the like are also used as diagnostic agents to detect SRIF receptors expressed in cancers. Two similar octapeptide analogs having 6-membered rings, i.e. Lanreotide and Vapreotide, have also been developed, see Smith-Jones et al., Endocrinology, 140, 5136-5148 (1999). A number of versions of these somatostatin analogs have been developed for use in radioimaging or as radiopharmaceuticals in radionuclide therapy. For radioimaging, for example, labeling with 123I can be used as disclosed in U.K. Patent Application 8927255.3 and as described in Bakker et al., 1991, J. Nucl. Med., 32:1184-1189. Proteins have been previously radiolabeled through the use of chelating agents, and there are various examples of complexing somatostatin analogs with 99Tc, 90Y or 111In, see U.S. Pat. Nos. 5,620,675 and 5,716,596. A variety of complexing agents have been used including DTPA; DOTA; HYNIC; and P2S2—COOH, U.S. Pat. No. 5,597,894 discloses analogs of Octreotide modified to facilitate radiolabeling.
Octreotide and other clinically used SRIF analogs interact significantly with three of the receptor subtypes, i.e. SSTR2, SSTR3 and SSTR5. SSTR2 and SSTR5 have recently been reported to mediate antiproliferative effects of SRIF on tumor cell growth; therefore, they may mediate the clinical effects of Octreotide in humans. U.S. Pat. No. 5,750,499 discloses SRIF analogs which are selective for SSTR1. A comprehensive review of SRIF and its receptors is found in Patel, Y. C. “Somatostatin and its receptor family”, Front. Neuroendocrinol, 1999, 20, 157-198.
SSTR4 was one of the later SRIF receptors cloned; it is not found in the abundance in normal human tissue as are some of the other receptors. It has high affinity for SRIF and SRIF-28, while it exhibits low affinity for many synthetic analogs of SRIF. In certain human tumors, SSTR4 mRNA may be the most frequently and most strongly expressed subtype receptor among the SST receptors. As a result of the many tumors that carry SRIF receptors, peptide radiopharmaceuticals have been developed for detection and visualization of such tumors and in addition, compounds that complex with 111In or 90Y are proving to be very promising radioligands for receptor-mediated radiotherapy.
Because of the presence of SSTR4 on some tumors, and because of the otherwise ubiquitous nature of the somatostatin receptors, it would be valuable to have somatostatin analogs that would bind strongly to SSTR4 while at the same time showing only minimal propensity for binding to the other 4 receptors. The search has continued for somatostatin analogs which are more potent than somatostatin and/or exhibit dissociated inhibitory functions, and particularly for analogs which are selective for SSTR4. Non-peptide SRIF agonists have been identified using combinatorial chemistry which exhibit selectivity for each of SSTR1 to SSTR5, Rohrer, S. P. et al., Science, 282, 737-740, 23 Oct. 1998. However, no peptide ligand has thus far been available that selectively binds to SSTR4 and exhibits fairly high affinity; as a result, efforts to determine the precise localization of SSTR4 in the body and to identify more of its biological actions have been hindered. Moreover, such lack of selective SSTR4 peptide ligands having relatively high affinity has hampered efforts to design more selective tumor diagnosis and treatment and radionuclide therapy, because only peptide ligands can be satisfactorily derivatized to incorporate complexing agents for radionuclides.